A recombinant lactobacillus that simultaneously expresses Clostridium perfringens α, β2, ε, β1 exotoxin and its construction method and application
A technology of Clostridium perfringens and Lactobacillus, which is applied in the field of veterinary medicine, can solve problems such as difficult to provide effective protection, and achieve the effect of preventing Clostridium perfringens infection
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Embodiment 1
[0034] Example 1 Construction of Recombinant Lactobacillus Simultaneously Expressing Clostridium perfringens α, β2, ε, β1 Exotoxin
[0035] 1. Detoxification transformation of Clostridium perfringens exotoxin
[0036]Through the bioinformatics analysis of Clostridium perfringens exotoxins α, β1, β2 and ε, the key gene loci that determine the toxicity of each exotoxin were determined, and the PCR method was used to detoxify and transform them into toxoids. Specifically: mutate residues 202-204 of the gene encoding α-toxin to GGC; mutate residues 699-702 of the gene encoding β-toxin (β1, β2) to GGA; The -318 residue is mutated to CCA, and the nucleotide sequences encoding the detoxified Clostridium perfringens exotoxin α, β2, ε, and β1 genes are shown in SEQ ID NO.5-8 respectively, and the encoded The amino acid sequences are shown in SEQ ID NO.1-4 respectively. Then, the PCR products of each toxin-encoding gene were cloned into pMD19-T vector sequentially, using BamH I and Kp...
Embodiment 2
[0042] Example 2 Recombinant lactobacillus immunogenicity detection
[0043] The present invention systematically evaluates the immunogenicity of the recombinant lactobacillus obtained in Example 1 by using BALB / c mice as animal models. The immunization procedure was as follows: the recombinant Lactobacillus pPG612-α-β2-ε-β1 / LP was inoculated by oral gavage, and the amount of bacteria in each BALB / c mouse was 2×10 8 CFU / mL, immunization once every 2 weeks, a total of 3 immunizations. At the same time, non-recombinant Lactobacillus (LP) and PBS were orally inoculated as controls. The levels of sIgA antibodies in feces, vaginal mucus and tears and the levels of IgG antibodies in serum were detected by ELISA method.
[0044] In addition, the challenge protection test was carried out on the immunized animals: the challenge (purified Clostridium perfringens exotoxin) test was carried out 15 days after the third immunization, and each mouse was intraperitoneally injected with 2 ti...
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